Establishment of an Optimized Co-transformation System of Common Wheat (Triticum aestivum) Using Particle Bombardment
HE Xiao-Lan1,2, WANG Jian-Wei1, CHEN Xin-Hong2,*, LI Wen-Xu3, TANG Hong1
1 School of Life and Health Science, Kaili University, Kaili 556011, China; 2 Shaanxi Key Laboratory of Genetic Engineering for Plant Breeding/College of Agronomy, Northwest A&F University, Yangling 712100, China; 3 Institute for Wheat Research, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
Abstract：Along with the increased commercialization and planting of a range of genetically modified (GM) crops globally, the biosafety of GM crops has become a hot topic and major public concern. To elucidate the optimal molar ratio for co-transformation and develop a safe, high-efficiency wheat (Triticum aestivum) transformation system, in this study, a combination of β-glucuronidase (gus) and a herbicide resistance gene (bar) was used to select stable transformants. Six independent co-transformation experiments were performed using either the minimal cassette Ubi-bar or the whole of pAHC20, with the Ubi-gus minimal cassette at different molar ratios. The whole pAHC25 plasmid was used as a reference, which was transferred into immature embryos of the wheat variety 'Kenong 199'. The results showed that the transient expression rate of gus gene increased significantly with the increase of Ubi-gus molar concentration, but there was no correlation between transient expression and stable expression. The transformation frequency differed significantly among combinations, in which the highest was obtained by combining Ubi-bar or pAHC20∶Ubi-gus at a molar ratio of 1∶2. The transformation frequency was significantly lower using the whole plasmid than the minimal constructs with co-transformation at a molar ratio of 1∶2, but significantly higher than that with 1∶1 and 1∶3. The stable integration, expression, and inheritance of the gus were confirmed by GUS assays and Southern blotting. The positive transgenic lines exhibited normal morphology except for poor seed set. This system may facilitate future transformation studies.
贺晓岚, 王建伟, 陈新宏, 李文旭, 汤宏. 基因枪介导的普通小麦共转化体系的优化[J]. 农业生物技术学报, 2021, 29(1): 146-158.
HE Xiao-Lan, WANG Jian-Wei, CHEN Xin-Hong, LI Wen-Xu, TANG Hong. Establishment of an Optimized Co-transformation System of Common Wheat (Triticum aestivum) Using Particle Bombardment. 农业生物技术学报, 2021, 29(1): 146-158.
[1] 叶兴国, 徐惠君, 杜丽璞, 等. 2014. 小麦规模化转基因技术体系构建及其应用[J]. 中国农业科学, 47(21): 4155-4171. (Ye X G, Xu H J, Du L P, et al.2014. Establishment and application of large-scale transformation systems in wheat[J]. Scientia Agricultura Sinica, 47(21): 4155-4171.) [2] 喻修道, 徐兆师, 陈明, 等. 2010. 小麦转基因技术研究及其应用[J]. 中国农业科学, 43(8): 1539-1553. (Yu X D, Xu Z S, Chen M, et al.2010. The progress and application of wheat transformation technology[J]. Scientia Agricultura Sinica, 43(8): 1539-1553.) [3] 张玮, 王静, 纪军, 等. 2011. 冬小麦新品种'科农199'选育和推广[J]. 中国生态农业学报, 19(5): 1215-1219. (Zhang W, Wang J, Ji J, et al.2011. Development of 'Kn199' new winter wheat variety and its cultivation in China[J]. Chinese Journal of Eco-Agriculture, 19(5): 1215-1219.) [4] 赵艳, 于彦春, 钱前, 等. 2003. 无载体主干序列的bar和cecropin B基因表达框共转化水稻[J]. 遗传学报, 30(2): 135-141. (Zhao Y, Yu Y C, Qian Q, et al.2003. Cotransformation of rice by bar and cecropin B gene expression cassettes lacking vector backbone sequences[J]. Acta Genetica Sinica, 30(2): 135-141.) [5] Agrawal P, Kohli A, Twyman R, et al.2005. Transformation of plants with multiple cassettes generates simple transgene integration patterns and high expression levels[J]. Molecular Breeding, 16(3): 247-260. [6] Blechl A E, Anderson O D.1996. Expression of a novel high-molecular-weight glutenin subunit gene in transgenic wheat[J]. Nature Biotechnology, 7(14): 875. [7] Charng Y, Li K, Tai H, et al.2008. An inducible transposon system to terminate the function of a selectable marker in transgenic plants[J]. Molecular Breeding, 21(3): 359-368. [8] Chen L, Marmey P, Taylor N J, et al.1998. Expression and inheritance of multiple transgenes in rice plants[J]. Nature Biotechnology, 16(11): 1060-1064. [9] Christou P, Ford T L, Kofron M.1991. Production of transgenic rice (Oryza Sativa L.) plants from agronomically important Indica and Japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos[J]. Bio-Technology, 9(10): 957-962. [10] Christou P, Ford T.1995. Parameters influencing stable transformation of rice immature embryos and recovery of transgenic plants usinge discharge particle acceleration[J]. Annals of Botany, 75(4): 407-413. [11] Dufourmantel N, Dubald M, Matringe M, et al.2007. Generation and characterization of soybean and marker-free tobacco plastid transformants over-expressing a bacterial 4-hydroxyphenylpyruvate dioxygenase which provides strong herbicide tolerance[J]. Plant Biotechnology Journal, 5(1): 118-133. [12] François I E J A, Broekaert W F, Cammue B P A.2002. Different approaches for multi-transgene-stacking in plants[J]. Plant Science, 163(2): 281-295. [13] Fu X, Fontana S, Bong B B, et al.2000. Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns[J]. Transgenic Research, 9(1): 11-19. [14] Gao C, Jiang L, Folling M, et al.2006. Generation of large numbers of transgenic kentucky bluegrass (Poa pratensis L.) plants following biolistic gene transfer[J]. Plant Cell Reports, 25(1): 19-25. [15] He Y, Jones H D, Chen S, et al.2010. Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv Stewart) with improved efficiency[J]. Journal of Experimental Botany, 61(6): 1567-1581. [16] Henry R J, Furtado A.2014. Cereal genomics: Methods and protocols[M]. Humana Press, New York, the USA, pp. 201-218. [17] Huang S, Gilbertson L, Adams T, et al.2004. Generation of marker-free transgenic maize by regular two-border Agrobacterium transformation vectors[J]. Transgenic Research, 13(5): 451-461. [18] Isaac P G.1994. Protocols for nucleic acid analysis by nonradioactive probes[M]. NJ: Humana Press, Totowa, the USA, pp. 9-15. [19] Jackson M A, Anderson D J, Birch R G.2013. Comparison of Agrobacterium and particle bombardment using whole plasmid or minimal cassette for production of high-expressing, low-copy transgenic plants[J]. Transgenic Research, 22(1): 143-151. [20] Khan R S, Nakamura I, Mii M.2011. Development of disease-resistant marker-free tomato by R/RS site-specific recombination[J]. Plant Cell Reports, 30(6): 1041-1053. [21] Kim J Y, Gallo M, Altpeter F.2012. Analysis of transgene integration and expression following biolistic transfer of different quantities of minimal expression cassette into sugarcane (Saccharum spp. hybrids)[J]. Plant Cell, Tissue and Organ Culture, 108(2): 297-302. [22] Kopertekh L, Broer I, Schiemann J.2009. Developmentally regulated site-specific marker gene excision in transgenic B. napus plants[J]. Plant Cell Reports, 28(7): 1075-1083. [23] Li J, Ye X, An B, et al.2012. Genetic transformation of wheat: Current status and future prospects[J]. Plant Biotechnology Reports, 6(3): 183-193. [24] Liu X, Jin W, Liu J, et al.2011. Transformation of wheat with the HMW-GS 1Bx14 gene without markers[J]. Russian Journal of Genetics, 47(2): 182-188. [25] Loc N T, Tinjuangjun P, Gatehouse A M, et al.2002. Linear transgene constructs lacking vector backbone sequences generate transgenic rice plants which accumulate higher levels of proteins conferring insect resistance[J]. Molecular Breeding, 9(4): 231-244. [26] Lowe B A, Prakash N S, Way M, et al.2009. Enhanced single copy integration events in corn via particle bombardment using low quantities of DNA[J]. Transgenic Research, 18(6): 831-840. [27] Matheka J M, Anami S, Gethi J, et al.2013. A new double right border binary vector for producing marker-free transgenic plants[J]. BMC research notes, 6(1): 448. [28] Miroshnichenko D, Filippov M, Babakov A, et al.2007. Genetic engineering of russian wheat genotypes for abiotic stress resistance[J]. Options Méditerranéennes, Series A, 12: 715-721. [29] Okubara P, Blechl A, Mccormick S, et al.2002. Engineering deoxynivalenol metabolism in wheat through the expression of a fungal trichothecene acetyltransferase gene[J]. Theoretical and Applied Genetics, 106(1): 74-83. [30] Prakash N S, Bhojaraja R, Shivbachan S K, et al.2009. Marker-free transgenic corn plant production through co-bombardment[J]. Plant Cell Reports, 28(11): 1655-1668. [31] Qin J B, Wang Y, Zhu C Q.2014. Biolistic transformation of wheat using the HMW-GS 1Dx5 gene without selectable markers[J]. Genetics and Molecular Research, 13(2): 4361-4371. [32] Rasco-Gaunt S, Riley A, Barcelo P, et al.1999. Analysis of particle bombardment parameters to optimise DNA delivery into wheat tissues[J]. Plant Cell Reports, 19(2): 118-127. [33] Rasco-Gaunt S, Riley A, Cannell M, et al.2001. Procedures allowing the transformation of a range of European elite wheat (Triticum aestivum L.) varieties via particle bombardment[J]. Journal of Experimental Botany, 52(357): 865-874. [34] Romano A, Raemakers K, Bernardi J, et al.2003. Transgene organisation in potato after particle bombardment-mediated (co-) transformation using plasmids and gene cassettes[J]. Transgenic Research, 12(4): 461-473. [35] Sandhu S, Altpeter F.2008. Co-integration, co-expression and inheritance of unlinked minimal transgene expression cassettes in an apomictic turf and forage grass (Paspalum notatum Flugge)[J]. Plant Cell Reports, 27(11): 1755-1765. [35] Sestili F, Janni M, Doherty A, et al.2010. Increasing the amylose content of durum wheat through silencing of the SBEIIa genes[J]. Bmc Plant Biology, 1(10): 144. [36] Shi N, He G, Li K, et al.2007. Transferring a gene expression cassette lacking the vector backbone sequences of the lAxl high molecular weight glutenin subunit into two Chinese hexaploid wheat genotypes[J]. Agricultural Sciences in China, 6(4): 381-390. [37] Shiva Prakash N, Bhojaraja R, Shivbachan S K, et al.2009. Marker-free transgenic corn plant production through co-bombardment[J]. Plant Cell Reports, 28(11): 1655-1668. [38] Song H, Ren X, Si J, et al.2008. Construction of marker-free GFP transgenic tobacco by Cre/lox site-specific recombination system[J]. Agricultural Sciences in China, 7(9): 1061-1070. [39] Sparks C A, Jones H D.2009. Biolistics transformation of wheat[J]. Methods in Molecular Biology, 478: 71-92. [40] Tee C S, Maziah M.2005. Optimization of biolistic bombardment parameters for Dendrobium sonia 17 calluses using GFP and GUS as the reporter system[J]. Plant Cell, Tissue and Organ Culture, 80(1): 77-89. [41] Tuteja N, Verma S, Sahoo R K, et al.2012. Recent advances in development of marker-free transgenic plants: Regulation and biosafety concern[J]. Journal of Biosciences, 37(1): 167-197. [42] Vasil V, Castillo A M, Fromm M E, et al.1992. Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus[J]. Nature Biotechnology, 10(6): 667-674. [43] Vianna G R, Albino M M C, Dias B B A, et al.2004. Fragment DNA as vector for genetic transformation of bean (Phaseolus vulgaris L.)[J]. Scientia Horticulturae, 99(3-4): 371-378. [44] Vidal J, Kikkert J, Donzelli B, et al.2006. Biolistic transformation of grapevine using minimal gene cassette technology[J]. Plant Cell Reports, 25(8): 807. [45] Wang D, Zhao Q, Zhu D, et al.2006. Particle-bombardment-mediated co-transformation of maize with a lysine rich protein gene (sb401) from potato[J]. Euphytica, 150(1-2): 75-85. [46] Wang G P, Yu X D, Sun Y W, et al.2016. Generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation[J]. Frontiers in Plant Science, 7: 1-16. [47] Wang K, Liu H, Du L, et al.2017. Generation of marker-free transgenic hexaploid wheat via an Agrobacterium-mediated co-transformation strategy in commercial Chinese wheat varieties[J]. Plant Biotechnology Journal, 15(5): 614-623. [48] Yao Q, Cong L, Chang J L, et al.2006. Low copy number gene transfer and stable expression in a commercial wheat cultivar via particle bombardment[J]. Journal of Experimental Botany, 57(14): 3737-3746. [49] Yao Q, Cong L, He G, et al.2007. Optimization of wheat co-transformation procedure with gene cassettes resulted in an improvement in transformation frequency[J]. Molecular Biology Reports, 34(1): 61-67. [50] Yoder J I, Goldsbrough A P.1994. Transformation systems for generating marker-free transgenic plants[J]. Bio/technology, 12(3): 263-267. [51] Zhang K, Liu J, Zhang Y, et al.2015. Biolistic genetic transformation of a wide range of Chinese elite wheat (Triticum aestivum L.) varieties[J]. Journal of Genetics and Genomics, 42(1): 39-4
